Development of the Efficient Electroporation Protocol for Leuconostoc mesenteroides

Area of Science:

• Food Biotechnology
• Microbiology
• Synthetic Biology

Background:

• *Leuconostoc mesenteroides* is crucial in food biotechnology for producing metabolites and exopolysaccharides.
• Genetic manipulation of *L. mesenteroides* is challenging due to inefficient DNA transfer, high nuclease activity, and restriction-modification systems.
• Existing electroporation methods yield inconsistent results, hindering metabolic engineering and synthetic biology.

Purpose of the Study:

• To develop and validate a reproducible electroporation protocol for *L. mesenteroides* strain H32-02 Ksu.
• To overcome specific barriers hindering genetic transformation in this strain.
• To enhance the strain's amenability to genetic engineering for biotechnological applications.

Main Methods:

• Optimized cell wall weakening and osmotic protection steps.
• Developed a gentle electrical stimulus for membrane permeabilization.
• Established recovery conditions to minimize cell death and DNA degradation.
• Matched plasmid DNA methylation patterns to the recipient's restriction profile.
• Selected an *E. coli* strain with a compatible methylation profile for plasmid production.

Main Results:

• Achieved a 40-fold increase in transformation efficiency compared to baseline.
• Maximized transformation efficiency at 8 × 10^2 CFU µg^-1 DNA.
• Demonstrated a 3.5-fold increase in transformant yield by optimizing plasmid source.
• Showed a 3-fold increase in transformant number by reducing pulse voltage.
• Provided consistent and reproducible access to transformants for the first time.

Conclusions:

• The developed protocol significantly enhances *L. mesenteroides* transformation efficiency and reproducibility.
• Strain-specific barriers in *Leuconostoc* can be overcome through optimized genetic manipulation techniques.
• This methodology provides a foundation for constructing improved strains for food bioprocessing and expressing heterologous enzymes.
• The principles may be transferable to other lactic acid bacteria with similar genetic manipulation challenges.